Recombinant technology provides an attractive method for producing proteins of high purity in large quantities. Conventional techniques for recombinantly producing proteins in eukaryotes require that the gene of interest be first inserted into an expression plasmid, in vitro, prior to transfection of a host cell. The expression plasmid includes a promoter, which allows RNA polymerase to specifically bind to the DNA sequence in order to initiate transcription. A polyadenylation signal may also be present. A selectable marker is used to allow the identification of host cells which express the gene of interest.
Standard techniques for transfecting host cells require the use of two separate expression cassettes, one bearing the gene of interest driven by an appropriate promoter, and the other including a selectable marker driven by a separate promoter. These cassettes may be present in a single plasmid or may be delivered to the host cell using separate vectors. However, such techniques may result in reduced recoveries of clones containing the gene of interest due to deletion or inactivation of the cassette expressing the same. Alternatively, if separate vectors are used, constructs including only the selectable marker and not the gene of interest, may be stably integrated into the host cell genome. Thus, background effects may be seen caused by false positive clones carrying only the marker plasmid and not the gene of interest.
More recently, expression constructs have been developed which include one or more selectable markers, in addition to the gene of interest, under the control of a single promoter. Such constructs are referred to in the art as "dicistronic" or "bicistronic." For example, constructs have been developed which include an internal ribosome entry site known as "IRES" derived from, e.g., the encephalomyocarditis virus (EMCV). The IRES element permits the translation of two or more open reading frames from a single messenger RNA, one encoding the recombinant protein of interest and the other encoding the selectable marker. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997 22 150-161.
Although the use of a single expression cassette decreases the number of false positives, the technique requires that the gene of interest be first subcloned into an expression cassette including a promoter, prior to use. If the expression of multiple genes is desired, such as from a cDNA library, the technique is particularly cumbersome, time-consuming and labor-intensive. Accordingly, there is a continued need for improved methods for producing proteins recombinantly.
Penolazzi et al., Anal. Biochem. (1997) 248:190-193, describe the direct transfection of PCR-generated DNA fragments, labeled with ethididum bromide (EtdBr), into mammalian cells. The cells were reported to retain the EtdBr-DNA for 48 hours.
Escher, D. and Schaffner, W., BioTechniques (1996) 21:848-854, describe a system where a linearized recipient vector containing a yeast GAL4 DNA binding domain (DBD) under the control of a CMV promoter, and an SV40 origin of replication, is cotransfected into a recipient mammalian cell with a fragmented DNA including a potential activation domain. The technique is said by the authors to produce functional fusion proteins by in vivo ligation of the components.
However, none of the aforementioned references describes a noncloning system whereby a promoter element, a gene of interest, and a selectable marker are individually cotransfected into a mammalian cell to result in expression of the protein encoded by the gene of interest.